Project Summary/Abstract The objective of this proposal is to unravel novel molecular mechanisms of regulation of serotonin (5-HT) receptors, which are cell surface proteins that transduce extracellular signals into intracellular signals. 5-HT receptors are prototypical G protein-coupled receptors (GPCRs), constitute a significant portion of the human genome. They participate in nearly every aspect of physiology and in many disease states. Nevertheless, our understanding of how these receptors transfer signals remains incomplete. In particular, many aspects of their intracellular trafficking, and the specific proteins involved in determining the fate of GPCRs, remain mysterious. This will be accomplished using the following methodologies: confocal microscopy, subcellular fractionation, biochemical methods, fusion proteins and transfections into -- or silencing of genes expressed in -- Hek293 cells. The hypothesis upon which this work is based is that two key protein families known to be involved in degradation of growth factor receptors - Cbl and HRS - are critical mediators of recycling of 5-HT2A receptors. The first aim focuses on c-Cbl, and the second on HRS. The work is significant in that our preliminary data demonstrate unexpected roles for c-Cbl and HRS in recycling (rather than degradation) of 5-HT2A receptors. The work is clinically relevant in that 5-HT regulates mood, aggression, anxiety, sexual behavior, sleep-wakefulness, vascular tone, immune function, cell growth and proliferation, and fluid and electrolyte homeostasis. This work is particularly germane to the Veteran population in that those disorders are frequently encountered in VA clinical settings. Importantly, the incidence and severity of many of those maladies will worsen as our Veteran population continues to age. The topic of this proposal also is significant because of the fundamental importance of understanding GPCR signal transduction as it relates to all functions of human cells. GPCRs comprise the largest known family of integral membrane receptor proteins. GPCRs represent key therapeutic targets for the treatment of many diseases of Veterans including hypertension, atherosclerosis, stroke, heart failure, cancer, asthma, renal diseases, diabetes mellitus, traumatic brain injury, psychiatric diseases, neurodegenerative diseases, and immune disorders including AIDS. Ultimately, the type of fundamental research proposed in this application will likely lead to new therapeutic insights and strategies that will form the basis for clinical interventions in multiple disease states. PUBLIC HEALTH RELEVANCE: Relevance of the Research to the VA Patient Care Mission The work covered in this proposal is relevant to the VA patient care mission in two respects. First, it will add to our knowledge about how 5-HT regulates mood, aggression, anxiety, sexual behavior, sleep-wakefulness, vascular tone, immune function, cell growth and proliferation, and fluid and electrolyte homeostasis. This work is particularly germane to the Veteran population in that anxiety, sleep disorders, depression, vascular headache, atherosclerotic-thrombotic vascular diseases and hypertension are frequently encountered in VA clinical settings. Importantly, the incidence and severity of many of those maladies will worsen as our Veteran population continues to age. The second reason why the topic of this proposal is significant involves the fundamental importance of understanding GPCR signal transduction as it relates to all functions of human cells. GPCRs comprise the largest known family of integral membrane receptor proteins. Recent estimates suggest that the GPCRs represent H1,000 members, and the GPCR family makes up >1% of the human genome. GPCRs respond to a wide variety of stimuli including hormones, neurotransmitters, light, odor, taste, and peptides. Consequently, they play critical roles in the regulation of all organ systems and hormonal functions. The central importance of GPCRs in human physiology and diseases is highlighted by the fact that more than half of all medications prescribed today are targeted at GPCRs. GPCRs represent key therapeutic targets for the treatment of many diseases of Veterans including hypertension, atherosclerosis, stroke, heart failure, cancer, asthma, renal diseases, diabetes mellitus, psychiatric diseases, neurodegenerative diseases, and immune disorders including AIDS. Ultimately, the type of fundamental research proposed in this application will likely lead to new therapeutic insights and strategies that will form the basis for clinical interventions in multiple disease states.